A high spin form of cytochrome P-448 highly purified from PCB-treated rats—II: Characteristic requirement of cytochrome b5 for maximum activity

doi:10.1016/0006-2952(83)90005-9

Biochemical Pharmacology

Volume 32, Issue 17, 1 September 1983, Pages 2479-2483

https://doi.org/10.1016/0006-2952(83)90005-9 Get rights and content

Abstract

A high spin form of cytochrome P-448 (PCB P-448-H), highly purified from microsomes of PCB-treated rats, catalyzed oxidations of several compounds and required cytochrome b₅ for its full activities in all oxidations examined. PCB P-448-H catalyzed the hydroxylation of aniline and O-dealkylations of p-alkoxy derivatives of aniline and nitrobenzene and 7-alkoxy derivatives of coumarin. Among the activities measured, hydroxylation of aniline and O-dealkylation of p-alkoxy derivatives of aniline were catalyzed by PCB P-448-H more efficiently than by PCB P-448-L, which was a low spin form of cytochrome P-448 purified from liver microsomes of PCB-treated rats. In all reactions, PCB P-448-H required cytochrome b₅ for maximum activity. Slight requirements were also seen with PCB P-448-L but varied equivocally depending on the substrates. Cytochrome b₅ showed its maximum effects on p-propoxyaniline O-depropylation activity at a molar ratio of cytochrome b₅ to PCB P-448-H of 1:2. The enhancement by cytochrome b₅ was more pronounced when lower concentrations of either the substrate or NADPH-cytochrome P-450 reductase were added to the reconstituted system. Based on these results, we confirm that PCB P-448-H is a unique form of cytochrome P-448 with respect to the requirements for cytochrome b₅ and is a good probe to study the mechanisms involved in the enhancement of drug oxidations by cytochrome b₅.

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Effects of cytochrome b₅ (b₅) on catalytic activities of human cytochrome P450 (CYP) 3A5, CYP3A4, and CYP3A7 coexpressed with human NADPH-cytochrome P450 reductase in Escherichia coli membranes were investigated using 14 substrates. The activities of CYP3A5 were enhanced by addition of b₅ in approximately one third of the substrates employed in this study. Such enhancement by b₅ was roughly similar to that of CYP3A4, while the activities of CYP3A7 were not enhanced by b₅ with any substrates employed. V_max values for midazolam 1′-hydroxylation and amitriptyline N-demethylation by CYP3A5 were increased about twice by addition of b₅, which was also seen with CYP3A4, although the extent of the effects of b₅ on S₅₀ (K_m) and Hill coefficient differed dependent on substrates used. In contrast, b₅ did not alter any of these kinetic parameters of CYP3A7. The effects of b₅ on kinetic parameters of CYP3A5 were similar to those of CYP3A4 but not CYP3A7. These results suggest that roles of b₅ in drug oxidation activities of CYP3A5 and CYP3A4 are different from those of CYP3A7.
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The conformation of cytochrome P-450 was studied in relation to the interaction with its substrate by the measurement of fluorescence energy transfer. Cytochrome P-450 I-c and cytochrome P-450 II-d, both obtained from the hepatic microsome of polychlorinated biphenyl-treated rats, were used as P-450 type and P-448 type, respectively, and benzo[a]pyrene and 7-ethoxycoumarin were used as substrate. The distance between the donor and acceptor, which was described by the Förster equation, was calculated on the basis of the fluorescence-energy transfer between the substrate as a donor and the heme of the enzyme as an acceptor. The distance was apparently changed by incorporation of the enzyme into phospholipid or by reduction of the heme, indicating that the treatments changed the conformation of the enzyme. Differences in the conformational change were observed between the two enzymes, and the conformational change of cytochrome P-450 II-d using benzo[a]pyrene as a substrate was different from that using 7-ethoxycoumarin. The results suggest that the substrate-binding sites of the two enzymes differ in position toward the heme, and that there are at least two different substrate sites in cytochrome P-450 II-d, one for benzo[a]pyrene and another for 7-ethoxycoumarin.
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Ten monoclonal antibodies reactive with a high spin form of rat cytochrome P-448 (P-448-H) were obtained from hybridoma clones established by a fusion between P3X63Ag8.653 mouse myeloma cells and spleen cells of a $BALB c$ mouse hyperimmunized with the cytochrome. One monoclonal antibody recognized an epitope characteristic for P-448-H. Five monoclonal antibodies were cross-reactive with a low spin form of rat cytochrome P-448, but not with cytochrome P-450. Reactivity of these monoclonal antibodies with microsomes of rats pretreated with drug metabolizing inducers and Western blots of the microsomal cytochrome P-450 components are also demonstrated.
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^☆: Portions of this work were presented at the Fifth International Symposium on Microsomes and Drug Oxidations, Tokyo, July 1981.

^†: To whom all correspondence should be ddressed at: Department of Pharmacology, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160, Japan.

^§: Present address: Department of Toxicology and Microbial Chemistry, Faculty of Pharmaceutical Sciences, Science University of Tokyo, Ichigaya, Shinjuku-ku, Tokyo 162, Japan.

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A high spin form of cytochrome P-448 highly purified from PCB-treated rats—II: Characteristic requirement of cytochrome b5 for maximum activity☆

Abstract

Archs Biochem. Biophys.

Biochem. biophys. Res. Commun.

Archs Biochem. Biophys.

Biochem. biophys. Res. Commun.

Biochem. biophys. Res. Commun.

Biochem. biophys. Res. Commun.

Biochem. biophys. Res. Commun.

J. biol. Chem.

J. biol. Chem.

Biochem. biophys. Res. Commun.

J. biol. Chem.

J. biol. Chem.

Analyt. Biochem.

Biochem. biophys. Res. Commun.

A high spin form of cytochrome P-448 highly purified from PCB-treated rats—II: Characteristic requirement of cytochrome b₅ for maximum activity☆